This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. Recent studies have focused on: 1) analysis of the solution conformation and dynamics of the ribonuclease H (RNase H) domain of HIV reverse transcriptase, and the characterization of substrate-induced active site formation; 2) understanding the protein-mediated interactions of RT substrates and inhibitors; 3) analysis of the conformational processes involved in going from p66 and other early forms of the polyprotein to formation of the mature heterodimer structure. Project 1. HIV-1 reverse transcriptase (RT), a critical enzyme of the HIV life cycle and an important drug target, undergoes complex and largely uncharacterized conformational rearrangements that underlie its asymmetric folding, dimerization and subunit-selective ribonuclease H domain (RH) proteolysis. During the past year, we reported the first in a series of studies that utilized a combination of NMR spectroscopy, small angle X-ray scattering and X-ray crystallography to characterize the p51 and p66 monomers and the conformational maturation of the p66/p66' homodimer. The p66 monomer exists as a loosely structured molecule in which the fingers/ palm/connection, thumb and RH substructures are connected by flexible (disordered) linking segments. The initially observed homodimer is asymmetric and includes two fully folded RH domains, while exhibiting other conformational features similar to that of the RT heterodimer. The RH' domain of the p66' subunit undergoes selective unfolding with time constant about 6.5 hours, consistent with destabilization due to residue transfer to the polymerase' domain on the p66' subunit. A simultaneous increase in the intensity of resonances near the random coil positions is characterized by a similar time constant. Consistent with the residue transfer hypothesis, a construct of the isolated RH domain lacking the two N-terminal residues is shown to exhibit reduced stability. These results demonstrate that RH' unfolding is coupled to homodimer formation. Project 2. Although the structure of the p66 monomer and the NMR studies performed on the p66/p66' homodimer provide insight into the early conformational rearrangements that occur in the homodimer, the low sensitivity of the NMR experiment precludes the direct observation of these processes. In order to fill this gap, and in collaboration with Dr. Lalith Perera of the computational core, we have performed molecular dynamics simulations on various RT domains in order to better understand the short term conformational rearrangements that can occur in the monomer. These studies are consistent with the various aspects of the model developed, in which a series of unimolecular rearrangements of the p66 monomer occurs which results in formation of a more open conformation, and this open conformation is then able to dimerize with other monomers that are present as a result of a conformational selection process. Some of the simulations targeted the fingers/palm subdomains. In the monomer, the angle between these two subdomains, defined using helices A in the fingers and F in the palm, is rather sharp, 45. After dissociation of the connection domain, simulations show that this angle can increase during the first few hundred nanoseconds of the simulation to values in the 70-100 range that are characteristic of the p66 subunit in the p66/p51 heterodimer. These calculations thus support the role of a unimolecular conformational rearrangement that is consistent with our conformational maturation model. Project 3. According to our recent studies, p66 monomers are able to form conformationally asymmetric p66/p66' homodimers that contains two, folded RH domains. The RH' domain on p66' is the subject to selective destabilization as a result of the transfer of residues from RH' to the polymerase' domain on p66'. We have modeled this destabilization by expressing a construct of the RH domain in which the two N-terminal residues are not present, and the third leucine residue is mutated to a methionine, RH&#8710;NT. Recent studies demonstrate that this construct is considerably less stable than the full RT domain, consistent with the model-based destabilization by residue transfer. During the past year, we performed hydrogen/deuterium exchange studies on both RH and the truncated construct, RH&#8710;NT. These studies are consistent with the importance of the N-terminal residues for domain stabilization, and more specifically, show that the amide exchange rate at the Phe440-Tyr441 cleavage site is greatly increased in the truncated construct.